U.S. patent application number 14/243388 was filed with the patent office on 2015-10-08 for use of polyenylpyrrole derivatives for treating inflammation.
This patent application is currently assigned to Yu-Chieh Lee. The applicant listed for this patent is Yu-Chieh LEE. Invention is credited to Ann CHEN, Kuo-Feng HUA, Shuk-Man KA, Yulin LAM, Sheau-Long LEE, Yu-Chieh LEE, Shih-Hsiung WU.
Application Number | 20150284355 14/243388 |
Document ID | / |
Family ID | 54209164 |
Filed Date | 2015-10-08 |
United States Patent
Application |
20150284355 |
Kind Code |
A1 |
HUA; Kuo-Feng ; et
al. |
October 8, 2015 |
USE OF POLYENYLPYRROLE DERIVATIVES FOR TREATING INFLAMMATION
Abstract
The preset invention relates to a method for treating
inflammation comprising administering a subject in need thereof
with a therapeutically effective amount of polyenylpyrrole
derivatives of formula (I) or a pharmaceutically acceptable salt
thereof.
Inventors: |
HUA; Kuo-Feng; (I-Lan,
TW) ; CHEN; Ann; (Taipei, TW) ; LAM;
Yulin; (Singapore, SG) ; WU; Shih-Hsiung;
(Taipei, TW) ; KA; Shuk-Man; (Taipei, TW) ;
LEE; Yu-Chieh; (Taoyuan County, TW) ; LEE;
Sheau-Long; (Taoyuan County, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LEE; Yu-Chieh |
Taoyuan County |
|
TW |
|
|
Assignee: |
Yu-Chieh Lee
Taoyuan County
TW
|
Family ID: |
54209164 |
Appl. No.: |
14/243388 |
Filed: |
April 2, 2014 |
Current U.S.
Class: |
514/422 ;
514/444; 514/460 |
Current CPC
Class: |
C07D 409/06 20130101;
A61K 31/35 20130101; C07D 309/38 20130101; C07D 405/06
20130101 |
International
Class: |
C07D 309/38 20060101
C07D309/38; C07D 409/06 20060101 C07D409/06; C07D 405/06 20060101
C07D405/06 |
Claims
1. A method for treating inflammation comprising administering a
subject in need thereof with a therapeutically effective amount of
a compound of formula (I): ##STR00005## wherein R.sup.1, R.sup.2
and R.sup.3 independently are H or an alkyl, and Ar is an aryl
group or a five-membered heteroaryl group containing one, two or
three heteroatoms selected from the group consisting of N, O and S,
wherein the aryl group and the heteroaryl group are unsubstituted
or substituted by one or two substituents independently selected
from the group consisting of halo and mesyl, provided that when
R.sup.3 is a methyl group, Ar is not a 3-chloropyrrolyl group, or a
pharmaceutically acceptable salt thereof.
2. The method of claim 1, wherein the aryl group is a 6-membered
monocyclic, 10-membered bicyclic, or 14-membered tricyclic aryl
group, being unsubstituted or substituted by one or two
substituents independently selected from the group consisting of
halo and mesyl.
3. The method of claim 3, wherein the aryl group is selected from
the group consisting of phenyl, naphthyl, pyrenyl, anthryl, and
phenanthryl, being unsubstituted or substituted by one or two
substituents independently selected from the group consisting of
halo and mesyl.
4. The method of claim 1, wherein the five-membered heteroaryl
group is selected from the group consisting of pyrrolyl, furanyl,
thiophenyl, oxazolyl, isoxazolyl, pyrazolyl, thiazolyl,
isothiazolyl, triazolyl, oxadiazolyl, and thiadiazolyl, being
unsubstituted or substituted by one or two substituents
independently selected from the group consisting of halo and
mesyl.
5. The method of claim 1, wherein R.sup.1 and R.sup.3 are
independently H or methyl, and R.sup.2 is H.
6. The method of claim 1, wherein Ar is selected from the group
consisting of 3-chloropyrrol-2-yl, 3-chlorothiophen-2-yl,
2-chlorophenyl, and 3-chloro-1-mesyl-pyrrol-2-yl.
7. The method of claim 1, wherein the compound of formula (I) is
selected from the group consisting of compound 1h, compound 1i,
compound 1j, compound 1k, compound 1l, compound 1m and 1n, having
the structures as follows: ##STR00006##
8. The method of claim 1, wherein the compound of formula (I) is
compound 1h, 1i or 1n, having the structures as follows:
##STR00007##
9. The method of claim 1, wherein the compound of formula (I) is in
a therapeutically effective amount to (i) inhibit production of
nitric oxide (NO) or expression of inducible nitric oxide synthase
(iNOS), interleukin-6 (IL-6) or tumor necrosis factor-.alpha.
(TNF-.alpha.), (ii) inhibit NLRP3 inflammasome-mediated IL-1.beta.
expression, or (iii) inhibit reactive oxygen species (ROS)
production, mitogen-activated protein kinase (MAPR)
phosphorylation, NF-.kappa.B activation or protein kinase C (PKC)
activation, in inflammatory cells of the subject.
10. The method of claim 9, wherein the inflammatory cells are
macrophages or dendritic cells.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a new use of
polyenylpyrrole derivatives for treatment of inflammation.
BACKGROUND OF THE INVENTION
[0002] Conjugated polyenes is an interesting class of widely
occurring natural polyketides as they have been shown to possess
excellent biological properties such as antibacterial (Shim S H et
al., (2011) J Nat Prod 74(3): 395-401), antifungal (Shim S H et al.
supra; Chomcheon P et al., (2010) Chemistry 16(36): 11178-11185)
and antitumor activities (Gross II et al. (2009) Org Lett 11(21):
5014-5017; Yang Y L et al. (2007) Chemistry 13(24): 6985-6991).
[0003] Inflammation occurs in response to numerous conditions
including physical injury, irritation, tumor growth in tissue, and
bacterial, parasitic, fungal, or viral infection. Inflammation
causes both local and systemic effects. Representative effects that
can occur at the site of injury, irritation, or disease are the
increase of vascular permeability, release of degradative enzymes
including metalloproteinase, migration of leukocytes to the
affected site, neutrophil burst response to destroy invading cells,
and the secretion of cytokines Important systemic effects include
pain, fever, and the acute response in the liver.
[0004] Inflammatory cells include lymphocytes, mononuclear
macrophages and dendritic cells. Once activated, these inflammatory
cells can induce a series of inflammatory responses by releasing
inflammatory mediators, against the infections or foreign
particles. Further, nitric oxide (NO), interleukin-6 (IL-6), and
TNF-.alpha. are important pro-inflammatory mediators that are
produced mainly by lipopolysaccharide (LPS)-activated macrophages
and mediate multiple biological effects, including the activation
of immune responses. Additionally, the inflammasome is a
multi-protein signal complex for activating caspase-1. Among the
inflammasome, the NLRP3 inflammasome is one of the most
well-studied. The NLRP3 inflammasome is activated by adenosine
triphosphate in LPS-activated macrophages, leading to caspase-1
activation and IL-1.beta. secretion (Hu Y, et al., J Immunol. 2010
Dec. 15; 185(12):7699-705).
[0005] In our previous works, we isolated certain polyketides from
thermophilic fungus Myceliophthora thermophila, and demonstrated
that some of these compounds exhibited antitumor activity (Yang Y L
et al. supra). We also synthesized a class of polyenylpyrroles and
their analogs which were evaluated for their anti-tumor activities.
However, the anti-inflammation activities of the polyenylpyrrole
derivatives are not disclosed.
BRIEF SUMMARY OF THE INVENTION
[0006] In this present invention, it is unexpectedly found that
polyenylpyrrole derivatives of formula (I) as defined herein
exhibit excellent anti-inflammatory activities. Particularly, these
compounds are non-cytotoxic to cells. Therefore, the present
invention provides a new approach for treatment of inflammation
with these polyenylpyrrole derivatives.
[0007] In one aspect, the invention provides a method for treatment
of inflammation comprising administering a subject in need thereof
with a therapeutically effective amount of a compound of formula
(I) or a pharmaceutically acceptable salt thereof:
##STR00001##
wherein R.sup.1, R.sup.2 and R.sup.3 independently are H or an
alkyl, and Ar is an aryl group or a five-membered heteroaryl group
containing one, two or three heteroatoms selected from the group
consisting of N, O and S, wherein the aryl group and the heteroaryl
group are unsubstituted or substituted by one or two substituents
independently selected from the group consisting of halo and mesyl,
provided that when R.sup.3 is a methyl group, Ar is not a
3-chloropyrrolyl group.
[0008] Also provided is use of the compound of formula (I) as
described herein in the manufacture of a medicament for treatment
of inflammation.
[0009] In certain embodiments of the invention, the aryl group is a
6-membered monocyclic, 10-membered bicyclic, or 14-membered
tricyclic aryl group. Examples of an aryl group include without
limitation phenyl, naphthyl, pyrenyl, anthryl, and phenanthryl.
[0010] In certain embodiments of the invention, the five-membered
heteroaryl group is selected from the group consisting of pyrrolyl,
furanyl, thiophenyl, oxazolyl, isoxazolyl, pyrazolyl, thiazolyl,
isothiazolyl, triazolyl, oxadiazolyl, and thiadiazolyl.
[0011] In one embodiment of the invention, R.sup.1 is H or
methyl.
[0012] In one embodiment of the invention, R.sup.3 is H or
methyl.
[0013] In one embodiment of the invention, R.sup.2 is H.
[0014] In some embodiments of the invention, Ar is selected from
the group consisting of 3-chloropyrrol-2-yl, 3-chlorothiophen-2-yl,
2-chlorophenyl, and 3-chloro-1-mesyl-pyrrol-2-yl.
[0015] In certain examples of the invention, the compound of
formula (I) is selected from the group consisting of compound 1h,
compound 1i, compound 1j, compound 1k, compound 1l, compound 1m and
1n, having the structures as follows
##STR00002##
[0016] In an embodiment of the invention, the compound of formula
(I) is in a therapeutically effective amount to (i) inhibit NO
production or iNOS, IL-6 or TNF-.alpha. expression, (ii) inhibit
NLRP3 inflammasome-mediated IL-1.beta. expression, or (iii) inhibit
reactive oxygen species (ROS) production, mitogen-activated protein
kinase (MAPR) phosphorylation, NF-.kappa.B activation or protein
kinase C (PKC) activation, in inflammatory cells of the
subject.
[0017] In an embodiment of the invention, the inflammatory cells
are macrophages or dendritic cells.
[0018] Also provided is a composition for use in treating
inflammation comprising the compound of formula (I) as described
herein. Further provided is use of a compound of formula (I) as
described herein for manufacture of a medicament for treating
inflammation. The details of one or more embodiments of the
invention are set forth in the description below. Other features or
advantages of the present invention will be apparent from the
following detailed description of several embodiments, and also
from the appending claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] For the purpose of illustrating the invention, there are
shown in the drawings embodiments. It should be understood,
however, that the invention is not limited to the preferred
embodiments shown. In the drawings:
[0020] FIG. 1 shows the backbone of the synthesized
polyenylpyrroles.
[0021] FIG. 2 shows the effect of polyenylpyrrole derivatives on
the expression of inflammatory mediators in RAW 264.7 macrophages.
In (A) and (C), cells (5.times.10.sup.5/ml) were incubated with
polyenylpyrrole derivatives or DMSO (vehicle) for 30 min followed
by stimulating with LPS (1 .mu.g/ml) for 24 h, then NO (A), IL-6
and TNF-.alpha. (C) concentration in culture medium were assayed by
Griess reaction and ELISA, respectively. In (B), cells
(5.times.10.sup.5/ml) were pretreated with polyenylpyrrole
derivatives or DMSO for 30 min followed by stimulating with LPS (1
.mu.g/ml) for 24 h, then the protein expression of iNOS and COX-2
were assayed by Western blot. In (A) and (C), the data are
expressed as the mean.+-.SD for three separate experiments, while
in (B), the results are representative of those obtained in three
different experiments and the histogram shows the quantification
expressed as the mean.+-.SD. *, **, and # indicate a significant
difference at the level of p<0.05, p<0.01, p<0.001
respectively compared to LPS group.
[0022] FIG. 3 shows the effect of compound 1h on the secretion of
IL-6 and TNF-.alpha. in J774A.1 macrophages, peritoneal
macrophages, and JAWSII dendritic cells. (A) J774A.1 macrophages
(4.times.10.sup.5/ml), (B) peritoneal macrophages
(4.times.10.sup.5/ml), and (C) JAWSII dendritic cells
(4.times.10.sup.5/ml) were incubated with compound 1h or DMSO for
30 min followed by stimulating with LPS (1 .mu.g/ml) for 24 h, then
IL-6 and TNF-.alpha. concentration in culture medium were assayed
by ELISA. The data are expressed as the mean.+-.SD for three
separate experiments. *, **, and *** indicate a significant
difference at the level of p<0.05, p<0.01, p<0.001
respectively compared to LPS group.
[0023] FIG. 4 shows the effect of compound 1h on NLRP3 inflammasome
activation in LPS+ATP-activated J774A.1 macrophages. (A) J774A.1
macrophages (1.times.10.sup.6/ml) and (B) peritoneal macrophages
(1.times.10.sup.5/ml) were incubated with compound 1h for 30 min
followed by LPS (1 .mu.g/ml) stimulating for 5.5 h, then the cells
were stimulated with ATP (5 mM) for an additional 30 min. The
IL-1.beta. in the culture medium was measured by ELISA and the
expression of active caspase-1 (p10) was measured by Western blot.
In (C) and (D), J774A.1 macrophages (1.times.10.sup.6/ml) were
incubated with LPS (1 .mu.g/ml) for 5.5 h, then cells were
incubated with compound 1h for 30 min followed by stimulating with
ATP (5 mM) for an additional 30 min. The IL-1.beta. and IL-6 in the
culture medium were measured by ELISA and the expression of active
caspase-1 (p10) was measured by Western blot. In (E), J774A.1
macrophages (1.times.10.sup.6/ml) were incubated with compound 1h
for 30 min followed by LPS (1 .mu.g/ml) stimulating for 6 h. The
expression of NLRP3 and proIL-1.beta. were measured by Western
blot. In ELISA, the data are expressed as the mean.+-.SD for three
separate experiments, while in Western blot, the results are
representative of those obtained in three different experiments and
the histogram shows the quantification expressed as the mean.+-.SD.
*, **, and *** indicate a significant difference at the level of
p<0.05, p<0.01, and p<0.001 respectively compared to
LPS+ATP or LPS group.
[0024] FIG. 5 shows the effect of compound 1h on ROS production and
MAPK phosphorylation in LPS-activated macrophages. In (A), RAW
264.7 macrophages (5 .times.10.sup.5/ml) were incubated with
compound 1h (20 .mu.M), N-acetyl cysteine (NAC; 10 mM) or DMSO
(vehicle) for 30 min followed by LPS (1 .mu.g/ml) stimulating for
the indicated time, then ROS levels were measured by detection of
the fluorescence intensity of the fluorophore carboxyl-DCF and
expressed relative to those at time zero. In (B), RAW 264.7
macrophages (5.times.10.sup.5/ml) were incubated with compound 1h
(20 .mu.M) or DMSO for 30 min followed by LPS (1 .mu.g/ml)
stimulating for 0-60 min, then the phosphorylation levels of
ERK1/2, JNK1/2, p38 were analyzed by Western blot. In (C), J774A.1
macrophages (5.times.10.sup.5/ml) were incubated with compound 1h
or DMSO for 30 min followed by LPS (1 .mu.g/ml) stimulating for 20
min, then the phosphorylation levels of ERK1/2, JNK1/2, p38 were
analyzed by Western blot. In (A), the data are expressed as the
mean.+-.SD for three separate experiments, while in (B) and (C),
the results are representative of those obtained in three different
experiments and the histogram shows the quantification expressed as
the mean.+-.SD. * indicates a significant difference at the level
of p<0.05 compared to LPS group.
[0025] FIG. 6 shows of compound 1h on NF-.kappa.B activation in
LPS-activated macrophages. (A) RAW 264.7 macrophages
(5.times.10.sup.5/ml) or (B) J774A.1 macrophages
(5.times.10.sup.5/ml) were incubated with compound 1h or DMSO for
30 min followed by LPS (1 .mu.g/ml) stimulating for 20 min, then
the phosphorylation levels of IKK-.alpha. and I.kappa.B-.alpha. and
total I.kappa.B-.alpha. protein level were analyzed by Western
blot. (C) RAW 264.7 macrophages (5.times.10.sup.5/ml) or (D)
J774A.1 macrophages (5.times.10.sup.5/ml) were incubated with
compound 1h (10-40 .mu.M) or DMSO for 30 min followed by LPS (1
.mu.g/ml) stimulating for 20 min, then the nuclear translocation of
NF-.kappa.B were analyzed by ELISA. (E) RAW-Blue.TM. cells
(5.times.10.sup.5/ml) were incubated with compound 1h (2.5-40
.mu.M) or DMSO for 30 min followed by LPS (1 .mu.g/ml) stimulating
for 24 h, then the SEAP activity was measured by QUANTI-Blue.TM..
In (A) and (B), the results are representative of those obtained in
three different experiments and the histogram shows the
quantification expressed as the mean.+-.SD. p-IKK-.alpha. and
p-I.kappa.B-.alpha. are normalized to IKK-.alpha. and actin
respectively. In (C), (D), and (E), the data are expressed as the
mean.+-.SD for three separate experiments. *, **, and *** indicate
a significant difference at the level of p<0.05, p<0.01, and
p<0.001 respectively compared to LPS group.
[0026] FIG. 7 shows the effect of compound 1h on ROS production and
PKC-.alpha. phosphorylation in ATP-activated macrophages. In (A),
J774A.1 macrophages (1.times.10.sup.6/ml) were incubated with LPS
(1 .mu.g/ml) for 5.5 h and then incubated with compound 1 h (20 M),
DPI (100 .mu.M), or DMSO (vehicle) for 30 min followed by ATP (5
mM) stimulating for the indicated time, then ROS levels were
measured by detection of the fluorescence intensity of the
fluorophore carboxyl-DCF and expressed relative to those at time
zero. In (B), J774A.1 macrophages (1.times.10.sup.6/ml) were
incubated with compound 1h (20 .mu.M), DPI (100 .mu.M), or DMSO
(vehicle) for 30 min followed by LPS (1 .mu.g/ml) stimulating for
5.5 h, then the cells were stimulated with ATP (5 mM) for the
indicated time. The ROS levels were measured by detection of the
fluorescence intensity of the fluorophore carboxyl-DCF and
expressed relative to those at time zero. In (C), LPS-primed
J774A.1 macrophages (1.times.10.sup.6/ml) were incubated with
compound 1h (20 .mu.M) or DMSO (vehicle) for 30 min followed by ATP
(5 mM) stimulating for 0-60 min, then the phosphorylation level of
PKC-.alpha. was analyzed by Western blot. In (A) and (B), the data
are expressed as the mean.+-.SD for three separate experiments,
while in (C), the results are representative of those obtained in
three different experiments and the histogram shows the
quantification expressed as the mean.+-.SD. * indicates a
significant difference at the level of p<0.05 compared to ATP
group.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The following description is merely intended to illustrate
various embodiments of the invention. As such, specific embodiments
or modifications discussed herein are not to be construed as
limitations to the scope of the invention. It will be apparent to
one skilled in the art that various changes or equivalents may be
made without departing from the scope of the invention.
[0028] In order to provide a clear and ready understanding of the
present invention, certain terms are first defined. Additional
definitions are set forth throughout the detailed description.
Unless defined otherwise, all technical and scientific terms used
herein have the same meanings as is commonly understood by one of
skill in the art to which this invention belongs.
[0029] As used herein, the articles "a" and "an" refer to one or
more than one (i.e., at least one) of the grammatical object of the
article. By way of example, "an element" means one element or more
than one element.
[0030] The table below shows the abbreviations for some
terminologies.
TABLE-US-00001 NO nitric oxide IL-6 interleukin-6 TNF-.alpha. tumor
necrosis factor-.alpha. IL-1.beta. interleukin-1.beta. LPS
lipopolysaccharide ATP adenosine triphosphate TLR toll-like
receptor COX-2 cyclooxygenase-2 ROS reactive oxygen species MAPK
mitogen-activated protein kinase PKC protein kinase C
[0031] Innate immunity is typically triggered by
pathogen-associated molecular patterns that are shared by groups of
different microbial pathogens which are recognized by toll-like
receptor (TLR) or other cellular receptor expressed on the cell
surface of immune cells (Medzhitov R, Janeway C A. (1997) Cell 91:
295-298). LPS is one kind of pathogen-associated molecular patterns
of gram-negative bacteria which is able to induce inflammatory
mediator expression including NO, TNF-.alpha., and IL-6 by binding
to TLR4 (Takeda K, Kaisho T, Akira S. (2003) Annu. Rev. Immunol 21:
335-376). Unlike other cytokines, IL-1.beta. is synthesized as an
inactive immature form (precursor of IL-1.beta., proIL-1.beta.) via
transcriptional activation in activated macrophages (Hsu H Y, Wen M
H. (2002) J Biol Chem 277(25): 22131-22139). IL-1.beta. release is
controlled by NLRP3 inflammasome, a multi-proteins complex
containing caspase-1 (Cassel S L, Joly S, Sutterwala F S. (2009)
Semin Immunol 21(4): 194-198; Jin C, Flavell R A. (2010) J Clin
Immunol 30(5): 628-631). NLRP3 inflammasome controls the disease
progression and inflammatory responses caused by infection
(Kanneganti T D et al. (2006) Nature 440(7081): 233-236; Allen I C
et al. (2009) Immunity 30(4): 556-565; Gross O et al. (2009) Nature
459(7245): 433-436), obesity (Vandanmagsar B et al. (2011) Nat Med
17(2): 179-188), cholesterol crystals, (Duewell P et al. (2010)
Nature 464(7293): 1357-1361), silica crystals (Hornung V et al.
(2008) Nat Immunol 9(8): 847-856), amyloid-beta (Halle A et al.
(2008) Nat Immunol 9(8): 857-865), and uric acid crystals etc.
(Martinon F et al. (2006) Nature 440(7081): 237-241). Recent
findings suggest that ROS regulates either NLRP3 inflammasome
activation or TLR4 signaling (Hsu H Y et al. (2010) Eur J Immunol
40(3): 616-619; Liao P C et al. (2010) J Agric Food Chem 58(19):
10445-10451) and inhibition of NLRP3 activation is one of the
therapeutic strategies for inflammatory related diseases (Tsai P Y
et al. (2011) Free Radic Biol Med 51(3): 744-754).
[0032] In this invention, it is unexpectedly found that the
polyenylpyrrole derivatives of formula (I) have anti-inflammatory
activities, particularly in terms of the effects in reducing NO
expression, and also reducing expression of iNOS, IL-6 or
TFN-.alpha., and inhibiting NLRP3 inflammasome-mediated IL-1.beta.
expression, but do not decrease the expression of COX-2. It is also
found that the underlying mechanisms for the anti-inflammatory
activities of the polyenylpyrrole derivatives of formula (I) of the
invention involves the decreasing ROS production, MAPK
phosphorylation, and NF-.kappa.B activation, as well as ATP-induced
ROS production and PKC-.alpha. phosphorylation.
[0033] Therefore, the invention provides a method for treatment of
inflammation comprising administering a subject in need thereof a
therapeutically effective amount of a compound of formula (I) or a
pharmaceutically acceptable salt thereof:
##STR00003##
wherein R.sup.1, R.sup.2 and R.sup.3 independently are H or an
alkyl, and Ar is an aryl group or a five-membered heteroaryl group
containing one, two or three heteroatoms selected from the group
consisting of N, O and S, wherein the aryl group and the heteroaryl
group are unsubstituted or substituted by one or two substituents
independently selected from the group consisting of halo and mesyl,
provided that when R.sup.3 is a methyl group, Ar is not a
3-chloropyrrolyl group.
[0034] Also provided is a composition for use in treating
inflammation comprising the compound of formula (I) as described
herein. Further provided is use of a compound of formula (I) as
described herein for manufacture of a medicament for treating
inflammation.
[0035] As used herein, the term "alkyl" refers to a straight or
branched monovalent hydrocarbon containing, unless otherwise
stated, 1-20 carbon atoms (e.g., C.sub.1-C.sub.10, C.sub.1-C.sub.8,
or C.sub.1-C.sub.4,). Examples of alkyl include, but are not
limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl,
and t-butyl.
[0036] As used herein, the term "aryl" refers to a monovalent
6-carbon monocyclic, 10-carbon bicyclic, 14-carbon tricyclic
aromatic ring system. Examples of aryl groups includes without
limitation phenyl, naphthyl, pyrenyl, anthryl, and phenanthryl. In
one example, the aryl group is phenyl. The term "heteroaryl" as
used herein refers to a five membered monocyclic aromatic ring
system having one, two or three heteroatoms such as N, O and/or S.
Examples of heteroaryl groups includes without limitation pyrrolyl,
furanyl, thiophenyl, oxazolyl, isoxazolyl, pyrazolyl, thiazolyl,
isothiazolyl, triazolyl, oxadiazolyl, and thiadiazolyl. Preferred
examples of heteroaryl groups are pyrrolyl and thiophenyl. The aryl
and heteroaryl groups are unsubstituted or substituted by one or
two substituents, including without limitation, halo or mesyl
(CH.sub.3SO.sub.2--).
[0037] In some embodiments of the invention, R.sup.1 and R.sup.3
are independently H or methyl, and R.sup.2 is H.
[0038] In some embodiments of the invention, Ar is selected from
the group consisting of 3-chloropyrrol-2-yl, 3-chlorothiophen-2-yl,
2-chlorophenyl, and 3-chloro-1-mesyl-pyrrol-2-yl.
[0039] Specifically, Table 1 shows exemplary compounds of formula I
of the invention.
TABLE-US-00002 TABLE 1 Example R.sup.1 R.sup.2 R.sup.3 Ar 1h H H H
3-chloropyrrol-2-yl 1i Me H H 3-chloropyrrol-2-yl 1j H H Me
3-chlorothiophen-2-yl 1k Me H Me 3-chlorothiophen-2-yl 1l Me H Me
2-chlorophenyl 1m Me H Me 3-chlorophenyl 1n Me H Me
3-chloro-1-mesyl-pyrrol-2-yl
[0040] The structures of the exemplified compounds are as
follows:
##STR00004##
[0041] As used herein, the term "treating" as used herein refers to
application or administration of one or more active agents to a
subject, who has a disease, a symptom of the disease or a
predisposition toward the disease, with the purpose to cure, heal,
alleviate, relieve, alter, remedy, ameliorate, improve, or affect
the disease, the symptom of the disease, or the predisposition
toward the disease. For example, the term "treating inflammation or
an inflammatory disorder" as used herein will refer to reducing
local or systemic inflammatory over-responses by inhibiting NO,
iNOS, IL-6 or TNF-.alpha. expression and inhibiting NLRP3
inflammasome-mediated IL-1.beta. expression, but does not decrease
the expression of COX-2.
[0042] The term "effective amount" as used herein refers to that
amount of an active agent or composition sufficient to achieve the
above-described therapeutic efficacies in a subject. The effective
amount may vary, for example, depending upon the types or dosage of
the agent or composition and the weight, age and healthy condition
of the subject to be treated.
[0043] As used herein, the term "subject" as used herein includes
human beings and animals, such as companion animals (e.g., dogs,
cats, and the like), farm animals (e.g., cows, sheep, pigs, horses,
and the like), or laboratory animals (e.g., rats, mice, guinea
pigs, and the like).
[0044] As used herein, the term "inflammatory disorder" as used
herein includes rheumatoid arthritis, systemic lupus erythematosus,
alopecia areata, ankylosing spondylitis, antiphospholipid syndrome,
autoimmune Addison's disease, autoimmune hemolytic anemia,
autoimmune hepatitis, autoimmune inner ear disease, autoimmune
lymphoproliferative syndrome (alps), autoimmune thrombocytopenic
purpura (ATP), Behcet's disease, bullous pemphigoid,
cardiomyopathy, celiac sprue-dermatitis, chronic fatigue syndrome
immune deficiency, syndrome (CFIDS), chronic inflammatory
demyelinating polyneuropathy, cicatricial pemphigoid, cold
agglutinin disease, Crest syndrome, Crohn's disease, Dego's
disease, dermatomyositis, juvenile dermatomyositis, discoid lupus,
essential mixed cryoglobulinemia, fibromyalgia-fibromyositis,
Grave's disease, Guillain-Barre, Hashimoto's thyroiditis,
idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura
(ITP), Iga nephropathy, insulin dependent diabetes (Type I),
juvenile arthritis, Meniere's disease, mixed connective tissue
disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris,
pernicious anemia, polyarteritis nodosa, polychondritis,
polyglancular syndromes, polymyalgia rheumatica, polymyositis and
dermatomyositis, primary agammaglobulinemia, primary biliary
cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome,
rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome,
stiff-man syndrome, Takayasu arteritis, temporal arteritis/giant
cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo,
and Wegener's granulomatosis.
[0045] The term "pharmaceutically acceptable salt" as used herein
refers to a salt which is not deleterious to the subject to be
treated and retain the biological effectiveness and properties of
the active compound. Salts may also be derived from the following
pharmacologically or physiologically acceptable inorganic and
organic acids: hydrochloric, hydrobromic, sulfuric, nitric,
fumaric, phosphoric, diphosphate, succinic, tartaric, acetic,
citric, methanesulfonic, formic, benzoic, and malonic, but not
being limited therein. Salts may also be derived from the following
pharmacologically or physiologically acceptable inorganic and
organic bases: alkali metal (e.g. sodium), alkaline earth metal
(e.g. magnesium), and ammonium salts, but not being limited
therein.
[0046] The compounds of the invention may be administered by a
medically acceptable route such as orally, parentally (e.g.
intramuscularly, intravenously, subcutaneously, interperitoneally),
transdermally, rectally, by inhalation and the like.
[0047] In addition, the compounds of the invention are preferably
formulated with a pharmaceutically acceptable carrier to form a
pharmaceutical composition for use in the above mentioned
treatments. Accordingly, the present invention further relates to a
pharmaceutical composition comprising at least one of the compounds
as described above or a pharmaceutically acceptable salt thereof
together with a pharmaceutically acceptable carrier, for use in the
treatment as described herein.
[0048] The carrier may serve as a diluent, vehicle, excipient, or
medium for the active ingredient. Some examples of suitable
excipients include saline, buffered saline, dextrose, water,
glycerol, ethanol, lactose, dextrose, sucrose, sorbitol, mannitol,
starches, gum acacia, calcium phosphate, alginates, tragacanth,
gelatin, calcium silicate, microcrystalline cellulose,
polyvinylpyrrolidone, cellulose, syrup, and methyl cellulose. The
composition can additionally include lubricating agents such as
talc, magnesium stearate, and mineral oil; wetting agents;
emulsifying and suspending agents; preserving agents such as
methyl- and propylhydroxy-benzoates; sweetening agents; and
flavoring agents.
[0049] The compositions of the invention can be in any forms as
desired, including but not limited to, tablets, pills, powders,
lozenges, sachets, cachets, suppositories, suspensions, emulsions,
solutions, syrups, soft and hard gelatin capsules, sterile
injectable solutions, packaged powders, mist, implants or patches.
The compositions of the invention may be prepared by conventional
methods known in the art of pharmacy.
[0050] Without further elaboration, it is believed the above
description has adequately enabled the present invention. The
following example is, therefore, to be construed as merely
illustrative, and does not limit of the remainder of the disclosure
in any way whatsoever. All of the publications, including patents,
cited herein are hereby incorporated by reference in their
entireties.
[0051] The present invention is further illustrated by the
following examples, which are provided for the purpose of
demonstration rather than limitation.
EXAMPLE
[0052] 1. Materials and Methods
[0053] 1.1 Materials
[0054] Based on the chemical structure of polyenylpyrroles,
auxarconjugatin A and 12E-isorumbrin, we synthesized a class of
polyenylpyrroles and their analogues. The 3-chloropyrrole group
plays an important role in the cytotoxicity effects of
auxarconjugatin A and 12E-isorumbrin; therefore, the
3-chloropyrrole is replaced with other 2- or 3-chlorosubstituted
aromatic rings. The different R positions of polyenylpyrroles are
replaced with H, Me, or n-Bu. The backbone of the synthesized
polyenylpyrroles was shown in FIG. 1 (Fang Z et al. (2010) J Med
Chem 53(22): 7967-7978).
[0055] LPS (from Escherichia coli 0111:B4), ATP, mouse antibodies
against mouse phospho-ERK1/2, phospho-JNK1/2, phospho-p38, and
actin were purchased from Sigma (St. Louis, Mo.). Rabbit antibodies
against mouse phospho-IKK-.alpha./.beta., IKK,
phospho-I.kappa.B-.alpha., I.kappa.B-.alpha., phospho-PKC-.alpha.,
IL-1.beta., caspase-1, iNOS, and COX-2, rabbit antibodies against
mouse phospho-IKK-.alpha./.beta., and horseradish
peroxidase-labeled second antibodies were obtained from Santa Cruz
Biotechnology (Santa Cruz, Calif. ). NLRP3 antibody was purchased
from Enzo Life Sciences, Inc. (Farmingdale, N.Y.). IL-1.beta.,
TNF-.alpha. and IL-6 ELISA kits were purchased from R&D Systems
(Minneapolis, Minn.).
[0056] 1.2 Cell Cultures
[0057] Murine macrophages RAW 264.7 and J774A.1 cells, and
immortalized C57BL/6 murine bone marrow-derived dendritic cell line
JAWSII, were purchased from American Type Culture Collection
(CRL-11904). RAW 264.7 macrophages stably transfected with the
NF-.kappa.B reporter gene (RAW-Blue.TM. cells) were purchased from
InvivoGen (San Diego, Calif.). RAW 264.7, J774A.1, and RAW-Blue.TM.
cells were grown in RPMI-1640 medium supplemented with 10%
heat-inactivated fetal bovine serum (FBS) (Life Technologies,
Carlsbad, Calif.). JAWSII cells were grown in RPMI-1640 medium
supplemented with 20% non-inactivated FBS and 5 ng/ml murine GM-CSF
(R&D Systems). All cells were cultured at 37.degree. C. in a 5%
CO.sub.2 incubator.
[0058] 1.3 AlamarBlue.RTM. Assay for Cell Viability
[0059] RAW 264.7 cells were seeded at a density of 5000 cells in
100 .mu.l RPMI 1640 medium with 10% heat-inactivated FBS per well
in 96-well flat-bottom plates and incubated for 24 h at 37.degree.
C. in a 5% CO.sub.2 incubator. Cells were incubated with tested
samples for 24 h and the AlamarBlue.RTM. assay was used to
determine the cytotoxicity of the test samples. The procedure was
conducted following the protocol described in the manufacture's
instruction (AbD Serotec Ltd).
[0060] 1.4 Enzyme-Linked Immunosorbent Assay (ELISA)
[0061] The effects of tested samples on IL-1.beta., TNF-.alpha. and
IL-6 production were measured by ELISA according to the
manufacturer's instruction. In brief, 50 .mu.l of biotinylated
antibody reagent and 50 .mu.l of supernatant were added to an
anti-mouse IL-1.beta., TNF-.alpha. and IL-6 precoated stripwell
plate, and incubated at room temperature for 2 h. After washing the
plate three times with washing buffer, 100 .mu.l of diluted
streptavidin-HRP concentrate was added to each well and incubated
at room temperature for 30 min. The washing process was repeated;
then 100 .mu.l of a premixed tetramethylbenzidine substrate
solution was added to each well and developed at room temperature
in the dark for 30 min. Following the addition of 100 .mu.l of stop
solution to each well to stop the reaction, the absorbance of the
plate was measured by a microplate reader at 450 nm wavelength.
[0062] 1.5 NO Inhibitory Assay
[0063] RAW 264.7 cells were seeded in 24-well plates at a density
of 5.times.10.sup.5 cells/ml, and then incubated with or without
LPS (1 .mu.g/ml) in the absence or presence of tested samples for
24 h. The effects of the tested samples on NO production were
measured indirectly by analysis of nitrite levels using the Griess
reaction.
[0064] 1.6 NF-.kappa.B Reporter Assay
[0065] RAW-Blue.TM. cells, RAW 264.7 macrophages which stably
express a secreted embryonic alkaline phosphatase (SEAP) gene
inducible by NF-.kappa.B, were seeded in 60 mm dishes at a density
of 5.times.10.sup.5 cells/ml, and grown overnight in a 5% CO.sub.2
incubator at 37.degree. C. Cells were pretreated with compound 1h
for 30 min followed by LPS stimulating for 24 h, and then the
medium was harvested. Medium (20 .mu.l) were then mixed with
QUANTI-Blue .TM. (InvivoGen) medium (200 .mu.l) in 96-well plates
at 37.degree. C. for 15 min. Results of the SEAP activity were
assessed by measuring the optical density at 655 nm using an ELISA
reader.
[0066] 1.7 Western Blot Assay
[0067] Whole cell lysates were separated by SDS-PAGE and
electrotransferred to a PVDF membrane. The membranes were incubated
in blocking solution--5% nonfat milk in phosphate buffered saline
with 0.1% Tween 20--at room temperature for 1 h. Each membrane was
incubated with a specific primary antibody at room temperature for
2 h. After washing three times in PBS with 0.1% Tween 20, the
membrane was incubated with an HRP-conjugated secondary antibody
directed against the primary antibody. The membrane was developed
by an enhanced chemiluminescence Western blot detection system.
[0068] 1.8 Measurement of Intracellular ROS Production
[0069] Intracellular ROS stimulated by LPS was measured by
detecting the fluorescence intensity of the
2',7'-dichlorofluorescein diacetate (H.sub.2DCFDA) oxidized product
(DCF) (Molecular Probes, Eugene, Oreg.). Briefly, for LPS-induced
ROS production, RAW 264.7 macrophages (5.times.10.sup.5/ml) were
grown in a phenol red-free RPMI medium for 24 h and then pretreated
with H.sub.2DCFDA (2 .mu.M), compound 1h (20 .mu.M), or NAC (10 mM)
at 37.degree. C. for 30 min followed by LPS stimulating for the
time as indicated. For ATP-induced ROS production, LPS-primed
J774A.1 macrophages (5.times.10.sup.5/ml) were grown in a phenol
red-free RPMI medium for 6 h and then pretreated with H.sub.2DCFDA
(2 .mu.M), compound 1h (20 .mu.M), or NAC (10 mM) at 37.degree. C.
for 30 min followed by ATP stimulating for the time as indicated.
The relative fluorescence intensity of fluorophore DCF, formed by
peroxide oxidation of the non-fluorescent precursor, was detected
at an excitation wavelength of 485 nm and an emission wavelength of
530 nm with a microplate absorbance reader (Bio-Rad Laboratories,
Inc).
[0070] 1.9 Measurement of NF-.kappa.B p65 Nuclear Translocation
[0071] Nuclear protein from RAW 264.7 and J774A.1 cells were
extracted using a Nuclear Extract Kit (Active Motif) according to
the manufacturer's instructions and nuclear NF-.kappa.B p65
activation quantified using an ELISA-based TransAM NF-.kappa.B kit
(Active Motif, Tokyo, Japan) according to the manufacturer's
protocol by reading the absorbance with a microplate absorbance
reader (Bio-Rad Laboratories, Inc) at 450 nm with a reference
wavelength of 655 nm
[0072] 1.10 Statistical Analysis
[0073] All values are given as mean.+-.SD. Data analysis involved
one-way ANOVA with a subsequent Scheffe test.
[0074] 2. Results
[0075] 2.1 Effect of polyenylpyrrole Derivatives on the Viability
of Macrophages
[0076] The aim of this study is to identify non-toxic
polyenylpyrrole derivatives which can be used as anti-inflammatory
agents. Compounds 1a-n were evaluated for their cytotoxicities
against the murine macrophages cell line RAW 264.7 after 24 h
treatment. As shown in Table 2, compounds 1a-g exhibited high
cytotoxicity against RAW 264.7 cells with IC.sub.50 below 10 .mu.M,
indicating that these compounds are not suitable for further
evaluation of their anti-inflammatory activities. The non-cytotoxic
compounds 1h-n exhibited anti-inflammatory activity by reducing NO
production in LPS-activated macrophages. The three most potent
compounds are 1h, 1i and 1n with ED.sub.50 values of 15.+-.2,
16.+-.2 and 17.+-.2 .mu.M respectively.
TABLE-US-00003 TABLE 2 Sample.sup.a R.sup.1 R.sup.2 R.sup.3 Ar
IC.sub.50 ED.sub.50 1a H H Me 3-chloropyrrol-2-yl .sup. <10
.mu.M.sup.b N.D. 1b Me H Me 3-chloropyrrol-2-yl <10 .mu.M N.D.
1c nBu H Me 3-chloropyrrol-2-yl <10 .mu.M N.D. 1d H Me Me
3-chloropyrrol-2-yl <10 .mu.M N.D. 1e Me Me Me
3-chloropyrrol-2-yl <10 .mu.M N.D. 1f Me Et Me
3-chloropyrrol-2-yl <10 .mu.M N.D. 1g nBu Me Me
3-chloropyrrol-2-yl <10 .mu.M N.D. 1h H H H 3-chloropyrrol-2-yl
>100 .mu.M 15 .+-. 2 .mu.M 1i Me H H 3-chloropyrrol-2-yl >100
.mu.M 16 .+-. 2 .mu.M 1j H H Me 3-chlorothiophen-2-yl >100 .mu.M
29 .+-. 6 .mu.M 1k Me H Me 3-chlorothiophen-2-yl >100 .mu.M 18
.+-. 2 .mu.M 1l Me H Me 2-chlorophenyl >100 .mu.M 18 .+-. 3
.mu.M 1m Me H Me 3-chlorophenyl >100 .mu.M 26 .+-. 3 .mu.M 1n Me
H Me 3-chloro-l-mesyl-pyrrol-2-yl >100 .mu.M 17 .+-. 2 .mu.M
.sup.aIC.sub.50 value expressed as the mean value of triplicate
wells from at least three experiments by AlamarBlue .RTM. assay.
.sup.bThe ED.sub.50 values that elicited a 50% inhibition of the
LPS-induced NO generation. N.D.: non-determined.
[0077] 2.2 Compounds 1h, 1i and 1n Decrease Production of NO, iNOS,
and IL-6 by LPS-Activated RAW 264.7 Macrophages
[0078] To investigate the inhibitory effect of compounds 1h, 1i and
1n on the LPS-induced inflammatory responses, the NO levels in the
supernatant of LPS-activated RAW 264.7 macrophages cultured with
DMSO (vehicle) or compounds 1h, 1i and 1n were measured by Griess
reaction. The experimental results indicated that treatment with
compounds 1h, 1i and 1n alone did not alter the background level of
NO (data not shown), but decreased the production of NO by
LPS-activated cells in a dose-dependent manner (FIG. 2A). We next
investigated the effect of compounds 1h, 1i and 1n on the protein
expression of iNOS, the NO producing enzyme. Treatment with
compounds 1h, 1i and 1n reduced the expression of iNOS protein when
compared with vehicle in LPS-activated RAW 264.7 macrophages, but
did not affect the COX-2 expression, an enzyme producing
prostaglandin E2 (FIG. 2B). In addition, we tested the effect of
compounds 1h, 1i and 1n on cytokine production by LPS-activated RAW
264.7 macrophages. We found that treatment with compounds 1h, 1i
and 1n alone did not alter the background level of IL-6 and
TNF-.alpha. in macrophages (data not shown), but decreased the
secretion of IL-6 by LPS-activated RAW 264.7 macrophages in a
dose-dependent manner and compound 1h is more potent than 1i and 1n
(FIG. 2C). The TNF-.alpha. secretion was slight reduced by
compounds 1h, 1i and 1n, but not significantly (FIG. 2C).
[0079] 2.3 Compound 1h Decreases Secretions of IL-6 and TNF-.alpha.
by LPS-Activated J774A.1 Macrophages, Peritoneal Macrophages, and
JAWSII Dendritic Cells
[0080] To confirm the anti-inflammatory activity of compound 1h,
the effect of compound 1h on LPS-induced cytokine secretion was
investigated using another murine macrophages cell line J774A.1 and
primary peritoneal macrophages from C57BL/6 mice. We found that
compound 1h reduced secretions of IL-6 and TNF-.alpha. in both
J774A.1 cells (FIG. 3A) and peritoneal macrophages (FIG. 3B).
Furthermore, compound 1h also reduced secretions of IL-6 and
TNF-.alpha. in murine dendritic cell line JAWSII cells (FIG.
3C).
[0081] 2.4 Compound 1h Reduces IL-1.beta. Secretion Through
Inhibiting NLRP3 Inflammasome
[0082] ATP is known to activate NLRP3 inflammasome in LPS-primed
macrophages which leads to caspase-1 activation and IL-1.beta.
secretion (Hu Y et al. (2010) J Immunol 185(12):
[0083] 7699-7705). To test whether compound 1h is able to modulate
NLRP3 inflammasome activation, a mouse macrophage cell line,
J774A.1, was selected (RAW 264.7 macrophages are not suitable for
studying NLRP3 inflammasome). The full activation of the NLRP3
inflammasome requires both a priming signal and an activation
signal, and therefore in the present study we investigate whether
compound 1h was able to modulate the priming signal and the
activation signal of NLRP3 inflammasome. Incubation of cells with
compound 1h before LPS and ATP treatment significantly inhibited
IL-1.beta. secretion and caspase-1 activation in a dose-dependent
manner (FIG. 4A). In the same condition, The IL-1.beta. inhibition
activity of compound 1h was confirmed in primary peritoneal
macrophages (FIG. 4B). In addition, to examine whether compound 1h
was able to affect the ATP-mediated activation signal, we incubated
LPS-primed macrophages with compound 1h for 30 min before ATP
stimulation and found that the compound 1h inhibited IL-1.beta.
secretion, but not caspase-1 activation (FIG. 4C), while compound
1h had no significant effect on IL-6 secretion (FIG. 4D). These
results indicated that compound 1h specifically inhibited NLRP3
inflammasome-mediated IL-1.beta. secretion, but not inhibited IL-6
secretion, which is independent of NLRP3 inflammasome. Furthermore,
we have tested compound 1h on its ability to inhibit NLRP3
expression (essential component of NLRP3 inflammasome) and
proIL-1.beta. (IL-1.beta. precursor) in LPS-activated cells. In
this experiment, cells were incubated with compound 1h for 30 min
followed by LPS stimulation for another 6 h. We found that compound
1h inhibited LPS-induced proIL-1.beta. expression in a
dose-dependent fashion, but increased NLRP3 expression (FIG.
4E).
[0084] 2.5 Compound 1h Inhibits ROS Production and MAPK Activation
in LPS-Activated Macrophages
[0085] ROS have been demonstrated to play important roles in
LPS-mediated cytokine expression (Hsu H Y, Wen M H. supra; Liao P C
et al., supra). To test whether compound 1h mediated
anti-inflammatory effect in LPS-activated cells through
down-regulation of ROS production, the intracellular ROS production
in LPS-activated RAW 264.7 macrophages was measured. We found that
LPS stimulation of cells rapidly induced ROS production and
pretreatment with N-acetyl cysteine (NAC), a potent antioxidant,
reduced ROS production. Compound 1h was able to reduce
LPS-stimulated ROS production, suggesting that the
anti-inflammatory effect of compound 1h could have been mediated
partially through its antioxidative activity (FIG. 5A).
[0086] LPS potently induces macrophage activation and the
production of pro-inflammatory cytokines by the activation of TLR4
through many signaling pathways, including the MAPK signaling
pathways (Su S C et al. (2006) Clin Chim Acta 374(1-2): 106-115).
To examine whether the effects of compound 1h on LPS-induced
macrophages are associated with MAPK signaling cascades, RAW 264.7
macrophages were treated with LPS in the presence or absence of
compound 1h. The phosphorylation levels of MAPK, including ERK1/2,
JNK1/2 and p38 were determined by Western blot analysis. The
experimental results showed that compound 1h inhibited the
phosphorylation levels of ERK1/2, JNK1/2, and p38 in LPS-activated
RAW 264.7 macrophages (FIG. 5B). These results were confirmed in
J774A.1 macrophages (FIG. 5C). These results indicate that compound
1h inhibits the activation of the MAPK signaling cascades in
LPS-activated macrophages.
[0087] 2.6 Compound 1h Inhibits NF-KB Activation in LPS-Activated
Macrophages
[0088] In resting macrophages, NF-KB is sequestered in the
cytoplasm as an inactive precursor complex by its inhibitory
protein, I.kappa.B. Upon LPS stimulation, I.kappa.B is
phosphorylated by I.kappa.B kinase (IKK), ubiquitinated, and
rapidly degraded via proteasomes to release NF-.kappa.B (Baeuerle P
A. (1998) Cell 95: 729-731). In determining whether compound 1h
could inhibit LPS-stimulated NF-.kappa.B signaling in macrophages,
we found that compound 1h inhibited the phosphorylation levels of
IKK-.alpha. and I.kappa.B-.alpha., and partially rescued
I.kappa.B-.alpha. degradation in LPS-activated RAW 264.7
macrophages (FIG. 6A) and J774A.1 macrophages (FIG. 6B). In
addition, compound 1h inhibited NF-.kappa.B nuclear translocation
in both RAW 264.7 (FIG. 6C) and J774A.1 macrophages (FIG. 6D).
Furthermore, by using NF-.kappa.B-dependent alkaline phosphatase
reporter cells, we demonstrated that NF-.kappa.B transcriptional
activity in LPS-stimulated macrophages was also reduced by compound
1h (FIG. 6E). These results indicate that compound 1h inhibits the
activation of the NF-.kappa.B signaling cascades in LPS-activated
macrophages.
[0089] 2.7 Compound 1h Inhibits ROS Production and PKC-.alpha.
Phosphorylation in ATP-Activated Macrophages
[0090] ATP induces ROS production through NADPH oxidase is required
for caspase-1 activation and IL-1.beta. secretion in macrophages
(Moore S F, MacKenzie A B. (2009) J Immunol 183(5): 3302-3308; Cruz
C M et al. (2007) J Biol Chem 282(5): 2871-2879). To determine
whether compound 1h-mediated IL-1.beta. down-regulation occurs via
the inhibition of ATP-induced ROS production, LPS-primed cells were
first incubated with compound 1h for 30 min before ATP stimulation.
We found that compound 1h reduces ATP-induced ROS production
slightly (FIG. 7A) but inhibited ATP-induced ROS production
significantly when it added before LPS priming (FIG. 7B). In
addition, we also found that compound 1h inhibited ATP-induced
PKC-.alpha. phosphorylation in LPS-primed cells (FIG. 7C).
[0091] In summary, we have identified compounds 1h-1n which were
able to inhibit LPS-induced NO production without reducing cell
viability. Among them, we further demonstrated compounds 1h, 1i and
1n decreased production of NO, iNOS and IL-6 in LPS activated
macrophages, and compound 1h inhibited NLRP3 inflammasome
activation as well as NO and IL-6 expression through the reduction
of both LPS- and ATP-induced ROS production and LPS-induced
activation of MAPK and NF-.kappa.B. The compounds of the invention,
particularly compound 1h, could be applicable for use in the
development of anti-inflammatory therapeutic.
[0092] It will be appreciated by those skilled in the art that
changes could be made to the embodiments described above without
departing from the broad inventive concept thereof. It is
understood, therefore, that this invention is not limited to the
particular embodiments disclosed, but it is intended to cover
modifications within the spirit and scope of the present invention
as defined by the appended claims.
[0093] From the above description, one skilled in the art can
easily ascertain the essential characteristics of the present
invention, and without departing from the spirit and scope thereof,
can make various changes and modifications of the invention to
adapt it to various usages and conditions. Thus, other embodiments
are also within the claims.
* * * * *